Certain agents such as tetanus toxoid can innately trigger the immune response, and may be administered in vaccines without modification. Other important agents are not immunogenic, however, and must be converted into immunogenic molecules or constructs before they can induce the immune response.
This invention relates generally to advantageous processes for making immunogenic constructs. The invention also relates to the resulting immunogenic constructs and vaccines prepared therefrom, and the use of such immunogenic constructs.
More specifically, the invention relates to methods of activating carbohydrate-containing antigens for use in preparing immunogenic constructs. Immunogenic constructs are very advantageously prepared by activating a carbohydrate-containing moiety with an organic cyanylating agent such as 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP).
A variety of cyanylating reagents are known per se, e.g., as reagents for activating insoluble particles to prepare gels for affinity chromatography. See Wilcheck et al., Affinity Chromatography. Meth. Enzymol., 104C:3-55. Wakelsman et al., J.C.S. Chem. Comm., 1976:21 (1976), reported that CDAP is a mild reagent that can be used for modifying protein cysteine groups. Kohn et al., Anal. Biochem, 115:375 (1981), compared CDAP, N-cyanotriethyl-ammonium tetrafluoroborate (CTEA), and p-nitrophenylcyanate (pNPC) as activating agents for agarose, an insoluble polysaccharide resin. Other researchers have used CDAP to activate other types of insoluble particles, such as Sepharose and glyceryl-controlled pore glass. See, e.g., Carpenter et al., Journal of Chromatography, 573:132-135 (1992).
U.S. Pat. No. 3,788,948 to Kagedal et al. generally describes a method that uses organic cyanate compounds to bind organic compounds containing a primary or secondary amino group to polymers containing one or more hydroxyl and/or primary and/or secondary amino groups, e.g., to bind water-soluble enzymes to water-insoluble polymers. Kagedal et al. describe a method using certain organic cyanate compounds such as pNPC having advantages over cyanogen bromide.
Similarly, Andersson et al., International Journal of Cancer, 47:439-444 (1991), report using CDAP to activate a soluble polysaccharide prior to conjugation with protein. They directly conjugated epidermal growth factor (EGF) to low molecular weight 40 kDa dextran activated with cyanate, and used very high dextran to EGF ratios of approximately 50:1 (wt./wt.) to produce dextran-EGF conjugates and studied the binding of this conjugate to cultured cells.
Kagedal et al. and Andersson et al., however, are not concerned with immunogenic constructs. Indeed, conjugates of proteins to low molecular weight dextrans have been reported to be poorly or non-immunogenic. T. E. Wileman, J. Pharm. Pharmacology, 38:264 (1985).
The degree of immunogenicity, of course, is an important property of immunogenic constructs for vaccination purposes. The process of vaccination employs the body's innate ability to protect itself against invading agents by immunizing the body with antigens that will not cause the disease but will stimulate the formation of antibodies, cells, and other factors that will protect against the disease. For example, dead organisms are injected to protect against bacterial diseases such as typhoid fever and whooping cough, toxoids are injected to protect against tetanus and diphtheria, and attenuated organisms are injected to protect against viral diseases such as poliomyelitis and measles.
It is not always possible, however, to stimulate antibody formation merely by injecting the foreign agent. The vaccine preparation must be immunogenic, that is, it must be able to induce an immune response. The immune response is a complex series of reactions that can generally be described as follows: (i) the antigen enters the body and encounters antigen-presenting cells that process the antigen and retain fragments of the antigen on their surfaces; (ii) the antigen fragments retained on the antigen-presenting cells are recognized by T cells that provide help to B cells; and (iii) the B cells are stimulated to proliferate and divide into antibody-forming cells that secrete antibodies against the antigen.
Antibodies to most bacterial polysaccharides have been shown to provide protection against infection with encapsulated bacteria. The inability of newborns and infants to mount vigorous responses to T-cell independent (TI) antigens, as exemplified by polysaccharides, has resulted in their extreme susceptibility to life-threatening infections with these organisms. This impaired immune response to TI antigens can be overcome by conjugating T-cell epitopes onto the polysaccharides, thereby converting them into T-cell dependent (TD) antigens.
There are two conjugation methods generally used for producing immunogenic polysaccharide constructs: (1) direct conjugation of carbohydrate and protein; and (2) indirect conjugation of carbohydrates and protein via a bifunctional linker or spacer reagent. Generally, both direct and indirect conjugation require chemical activation of the carbohydrate moiety prior to its derivatization.
Chemical activation refers to the conversion of a functional group to a form that can undergo additional chemical reactions, e.g., the addition of a functional group or of a large moiety such as a protein. Derivatization is the addition of functional chemical group(s) or spacer reagent(s) to a protein.
Unfortunately, artisans have encountered a number of problems in forming immunogenic constructs via conjugation using activation methods. For example, the production of conjugate vaccines has been a formidable challenge, in part, because of the difficulty in activating the polysaccharide and conjugating the protein under conditions that do not lead to their degradation or to the destruction of their immunogenic epitopes. In preparing immunogenic constructs, the method used should be sufficiently gentle to retain important antigenic sites, i.e., epitopes, on the molecules. Thus, it is desirable to maintain the integrity of the structure and to preserve epitopes in these compounds. Unfortunately, the preparation steps currently used in the art are frequently not gentle and can destroy native carbohydrate and/or protein structures.
Moreover, many of the known techniques for carbohydrate modification require anhydrous conditions. Unfortunately, however, carbohydrates are frequently insoluble in organic solvents. Marburg et al., J. Amer. Chem. Soc., 108:5282 (1986).
Thus, although there is a large body of chemical literature describing the modification of carbohydrates, much of it is unsuitable for use with aqueous-based antigens. One approach has been the modification of polysaccharides to enhance their solubility in organic solvents. For example, by replacing the acidic hydrogen on certain acidic polysaccharides with the hydrophobic tetrabutyl ammonium counter-ion, Marburg et al. were able to solubilize polysaccharides in organic solvents and activate hydroxyls with carbonyl diimidazole, a reagent which must be used in dry solvent. This method is used with polysaccharides, such as Haemophilus influenzae PRP and Pneumococcal polysaccharides type 6B and 19F. Coupling of proteins can also be achieved through reductive amination, either using the aldehyde found on the reducing end of the polysaccharide or created by oxidation of the carbohydrate. Both of these approaches have intrinsic limitations and, thus, for high molecular weight polysaccharides, coupling through the reducing end is usually slow and inefficient and oxidation often results in cleavage of the polysaccharide chain or otherwise affects the antigen.
Certain carbohydrates contain groups, such as amino or carboxyl groups, that can be more easily activated or derivatized before conjugation. For instance, the amino groups in Pseudomonas Fisher Type I can be easily derivatized with iodoacetyl groups and bound to a thiolated protein. The carboxyl groups in carbohydrates such as Pneumonococcal type III can be easily activated with water-soluble carbodiimides, such as EDC, and can then be coupled directly to protein. Unfortunately, however, this group of carbohydrates is limited.
Other carbohydrates have aldehyde groups at the terminal reducing end that can be exploited for derivatization and conjugation. It is also possible to create aldehyde groups with oxidizing reagents, e.g., sodium periodate. Aldehyde groups can be condensed with amino groups on protein or with a bifunctional linker reagent. This condensation reaction, especially with the terminal reducing end of a high molecular weight polysaccharide, however, often proceeds quite slowly and inefficiently. This is exacerbated when directly conjugating carbohydrate aldehydes to proteins. Thus, yields are often very low using this method. Moreover, sodium periodate may break up carbohydrates into smaller fragments and/or disrupt epitopes, which may be undesirable.
Most carbohydrates must be activated before conjugation, and cyanogen bromide is frequently the activating agent of choice. See, e.g., Chu et al., Inf. & Imm., 40:245 (1983), and Dick & Beurret, "Glycoconjugates of Bacterial Carbohydrate Antigens," Conjugate Vaccines, J. M. Cruse & R. E. Lewis (eds.), vol. 10, 48-114 (1989). The first licensed conjugate vaccine was prepared with CNBr to activate HIB PRP, which was then derivatized with adipic dihydrazide and coupled to tetanus toxoid using a water-soluble carbodiimide.
To briefly summarize the CNBr-activation method, cyanogen bromide is reacted with the carbohydrate at a high pH, typically a pH of 10 to 12. At this high pH, cyanate esters are formed with the hydroxyl groups of the carbohydrate. These, in turn, are reacted with a bifunctional reagent, commonly a diamine or a dihydrazide. These derivatized carbohydrates may then be conjugated via the bifunctional group. In certain limited cases, the cyanate esters may also be directly reacted to protein.
The high pH is necessary to ionize the hydroxyl group because the reaction requires the nucleophilic attack of the hydroxyl ion on the cyanate ion (CN.sup.-). As a result, cyanogen bromide produces many side reactions, some of which add neo-antigens to the polysaccharides. M. Wilcheck et al., Affinity Chromatography. Meth. Enzymol., 104C:3-55. More importantly, many carbohydrates or moieties such as HIB PRP and Pn6 can be hydrolyzed or damaged by the high pH necessary to perform the cyanogen bromide activation.
Another problem with the CNBr-activation method is that the cyanate ester formed is unstable at high pH and rapidly hydrolyzes, reducing the yield of derivatized carbohydrate and, hence, the overall yield of carbohydrate conjugated to protein. Many other nonproductive side reactions, such as those producing carbamates and linear imidocarbonates, are promoted by the high pH. Kohn et al., Anal. Biochem, 115:375 (1981). Moreover, cyanogen bromide itself is highly unstable and spontaneously hydrolyzes at high pH, further reducing the overall yield.
Furthermore, the cyanogen bromide activation is difficult to perform and unreliable. Cyanogen bromide is highly toxic and potentially explosive. Extreme care must be used when working with large quantities as used in manufacture. All operations must be carried out in a suitable fumehood. It is also known to those in the art that the activation is not easily reproducible because some batches of cyanogen bromide work well and some do not. Cyanogen bromide is also poorly soluble in water, making it difficult to control the amount of soluble cyanogen bromide available to react with the carbohydrate. Even use of the same batch of cyanogen bromide and apparently identical reaction conditions do not always lead to identical results.
In addition to these disadvantages, it is very difficult to control the degree of carbohydrate activation achieved by using cyanogen bromide. It is also very difficult to achieve a high level of carbohydrate activation using this method. Increasing the amount of cyanogen bromide present is ineffective and only leads to increased side reactions without an increase in activation. Kohn et al., Applied Biochem and Biotech, 9:285 (1984).
Thus, while cyanogen bromide activation has proven to be a very useful reagent, it has a number of limitations. For example, cyanogen bromide requires a high pH (10-12) in order to make the hydroxyls sufficiently nucleophilic to react with the cyanate ion. However, neither CNBr nor the cyanate ester intermediate is stable at high pH, and consequently most of the reagent either hydrolyzes or undergoes nonproductive or unwanted side reactions. Thus, the efficiency of polysaccharide activation is low. Furthermore, the high pH required for activation can hydrolyze or damage many pH-sensitive polysaccharides. In addition, CNBr is toxic and difficult to work with in small quantities.
Moreover, as noted above, other conjugation methods suffer from various drawbacks. For example, although polysaccharides such as Cryptococcus neoformans and Pneumococcal polysaccharide type 3 and VI antigen have carboxyl groups that can be activated with carbodiimides in preparation for coupling to a protein, and polysaccharides such as Pseudomonas Fisher type III have amino groups that can be conveniently used, these antigens form a relatively limited group of all polysaccharides. Other approaches are therefore needed to activate or functionalize the majority of polysaccharides.
Thus, there is a need in the art for a method to produce immunogenic constructs that is gentle, maintains the integrity of the structure of the carbohydrates and proteins, preserves epitopes in the compounds, is easy to perform, is reliable, is readily reproducible, is readily scaled up, and works with a wide variety of polysaccharides.